|
| Status |
Public on Aug 29, 2018 |
| Title |
WT_replicate_3 |
| Sample type |
RNA |
| |
|
| Source name |
WT
|
| Organism |
Drosophila melanogaster |
| Characteristics |
strain background: W1118
genotype/variation: TOP3B wildtype
age: Adult
tissue: head
|
| Growth protocol |
Drosophila melanogaster (D. melanogaster) flies were cultured on corn syrup-soy recipe food from Archon Scientific at 25±1°C and 60±5% humidity, under a 12h/12hr light/dark cycle. Wild-type W1118 flies were purchased from Bloomington Drosophila Stock Center, while Top3b p-element remobilized mutants Top3b26 was a generous gift from T.S. Hsieh.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
A total 200 microL of Drosophila heads/per sample were collected by fast freezing and sieving. Total RNA was extracted using TRIzol reagent according to the manufacturer's protocol (ThermoFisher, Waltham, MA USA). Three sets of independently prepared RNA samples employed, and the quality and quantity of RNA was assessed by NanoDrop (ThermoFisher) and by Agilent Bioanalyzer RNA 6000 Chip (Agilent, Santa Clara, CA) analysis.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng total RNA using the Low-Input Quick Amp Labeling Kit (Agilent, Santa Clara, CA) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were evaluated with the NanoDrop ND-1000 Spectrophotometer (Wilmington, DE).
|
| |
|
| Hybridization protocol |
A total of 825 ng Cy3-labeled cRNA (specific activity >6 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 microL containing Agilent fragmentation buffer and Agilent gene expression blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 microL of 2× Hi-RPM Hybridization Buffer was added to the fragmentation mixture and hybridized to Agilent D. melanogaster 4×44K Gene Expression Microarray for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were rinsed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute at 37°C with GE Wash buffer 2 (Agilent), and dried by slowly removing from Wash buffer 2.
|
| Scan protocol |
Following posthybridization rinses, arrays were scanned using an Agilent SureScan microarray Scanner at 5 micron, and hybridization intensity data extracted from the scanned images using Agilent’s Feature Extraction Software 9.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| Description |
W1118_III
|
| Data processing |
Raw microarray hybridization intensity data were log-transformed. Raw microarray data were log transformed to yield z-scores. The z-ratio was calculated as the difference between the observed gene z-scores for the experimental and the control comparisons, and dividing by the standard deviation associated with the distribution of these differences. Z-ratio values of ± 1.5 were used as cut-off values and calculated using a 5% false discovery rate (FDR) threshold. The complete set was tested for gene set enrichment using parametric analysis of gene set enrichment. For each pathway z-score, a p-value was computed using JMP 6.0 software to test for the statistical significance.
|
| |
|
| Submission date |
Aug 29, 2018 |
| Last update date |
Jun 22, 2020 |
| Contact name |
Supriyo De |
| Organization name |
NIA-IRP, NIH
|
| Department |
Laboratory of Genetics and Genomics
|
| Lab |
Computational Biology & Genomics Core
|
| Street address |
251 Bayview Blvd
|
| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21224 |
| Country |
USA |
| |
|
| Platform ID |
GPL17080 |
| Series (1) |
| GSE119185 |
Topoisomerase 3B Interacts with RNAi Machinery to Promote Heterochromatin Formation and Transcriptional Silencing |
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